Stages Of Parasite Life Cycle Research Articles

Discovery of GAMA, a Plasmodium falciparum Merozoite Micronemal Protein, as a Novel Blood-Stage Vaccine Candidate Antigen

One of the solutions for reducing the global mortality and morbidity due to malaria is multivalent vaccines comprising antigens of several life cycle stages of the malarial parasite. Hence, there is a need for supplementing the current set of malaria vaccine candidate antigens. Here, we aimed to characterize glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (GAMA) encoded by the PF08_0008 gene in Plasmodium falciparum. Antibodies were raised against recombinant GAMA synthesized by using a wheat germ cell-free system. Immunoelectron microscopy demonstrated for the first time that GAMA is a microneme protein of the merozoite. Erythrocyte binding assays revealed that GAMA possesses an erythrocyte binding epitope in the C-terminal region and it binds a nonsialylated protein receptor on human erythrocytes. Growth inhibition assays revealed that anti-GAMA antibodies can inhibit P. falciparum invasion in a dose-dependent manner and GAMA plays a role in the sialic acid (SA)-independent invasion pathway. Anti-GAMA antibodies in combination with anti-erythrocyte binding antigen 175 exhibited a significantly higher level of invasion inhibition, supporting the rationale that targeting of both SA-dependent and SA-independent ligands/pathways is better than targeting either of them alone. Human sera collected from areas of malaria endemicity in Mali and Thailand recognized GAMA. Since GAMA in P. falciparum is refractory to gene knockout attempts, it is essential to parasite invasion. Overall, our study indicates that GAMA is a novel blood-stage vaccine candidate antigen.

Keeping the clock set under the midnight sun: diurnal periodicity and synchrony of avian Isospora parasites cycle in the High Arctic

For Isospora (Protozoa: Eimeriidae) parasites of passerine birds, diurnal periodicity of oocyst output is a well-described phenomenon. From the temporal zone to the tropics, oocyst production is correlated with the light-dark cycle, peaking in the afternoon hours. However, nothing is known about the existence of diurnal periodicity of these parasites in the birds of High Arctic environments, under permanent light during summer. We sampled free-ranging Snow Bunting (Aves: Passeriformes), on Svalbard in summer and tested oocysts output of Isospora plectrophenaxia. Here we show that under the permanent light conditions of Arctic summer in the wild, Isospora plectrophenaxia, a parasite of the Snow Bunting, still keeps the 24-h rhythm of oocyst output with the peak in the post-meridiem hours, despite the absence of diurnal periodicity in host's activity. Our findings prove the ability of avian Isospora to invoke alternative cues for synchronizing the circadian rhythms. Possible cues and adaptive significance of diurnal periodicity of parasite output in High Arctic are discussed. The maintenance of synchronization and timing of the parasite life-cycle stages is under positive selection pressure even in permanent daylight in the Arctic.

Efficacy of Lasia spinosa leaf extract in treating mice infected with Trichinella spiralis

Trichinellosis is a widespread zoonoses for which no effective drug treatment is available at this time. Though anthelmintics such as mebendazole and albendazole are commonly used to treat human trichinellosis, none of these drugs are fully effective against the encysted or new-born larvae of Trichinella spiralis. In recent years, there has been a growing interest in developing newer anthelminthics from medicinal plants, particularly the ones used in traditional medicines in many parts of the world, due to the increasing spread of anthelminthic resistance and/or decreasing activity against encapsulated larval stages of parasites. The aim of the present study was to investigate the efficacy of leaf extract of Lasia spinosa (Araceae) against different life cycle stages of T. spiralis, i.e. adult (days3 and 4 post-infection), migrating larvae (days8, 9 and 10 post-infection) and encysted muscle larvae (days31-37 post-infection). The study showed that L. spinosa leaf extract is effective against all the three life cycle stages of parasite. Against the adult stage, an oral administration of plant extract at 800mg/kg dose revealed a 75.30% reduction in the number of adult worms, as compared to untreated controls at day10 post-infection. Whereas against migrating larvae, the same dose of plant extract given for 3days, reduced the number of larvae recovered from musculature of treated animals by 72.23%. However, in comparison of preceding two stages, the extract showed comparatively less efficacy against the encysted larvae of parasite. In this case, the 800mg/kg dose of extract given for 7days (after 30day of post-infection) revealed only 64.84% reduction in the number of encysted larvae, as was evident from larval count on day49 post-infection. Therefore, the results of this study indicate that leaf extract of L. spinosa possesses significant anthelminthic efficacy against the adult stages and migrating larvae of T. spiralis. On the other hand, the encysted muscle larvae of parasite are comparatively less sensitive to L. spinosa leaf extract treatment.

Identification of Echinococcus granulosus microRNAs and their expression in different life cycle stages and parasite genotypes

The aetiological agent of cystic hydatid disease, the platyhelminth parasite Echinococcus granulosus, undergoes a series of metamorphic events during its complex life cycle. One of its developmental stages, the protoscolex, shows a remarkable degree of heterogeneous morphogenesis, being able to develop either into the vesicular or strobilar direction. Another level of complexity is added by the existence of genotypes or strains that differ in the range of intermediate hosts where they can develop and form fertile cysts. These features make E. granulosus an interesting model for developmental studies. Hence, we focused on the study of the regulation of gene expression by microRNAs (miRNAs), one of the key mechanisms that control development in metazoans and plants and which has not been analysed in E. granulosus yet. In this study, we cloned 38 distinct miRNAs, including four candidate new miRNAs that seem to be specific to Echinococcus spp. Thirty-four cloned sequences were orthologous to miRNAs already described in other organisms and were grouped in 16 metazoan miRNA families, some of them known for their role in the development of other organisms. The expression of some of the cloned miRNAs differs according to the parasite life cycle stage analysed, showing differential developmental expression. We did not detect differences in the expression of the analysed miRNAs between protoscoleces of two parasite genotypes. This work sets the scene for the study of gene regulation mediated by miRNAs in E. granulosus and provides a new approach to study the molecules involved in its developmental plasticity and intermediate host specificity. Understanding the developmental processes of E. granulosus may help to find new strategies for the control of cystic hydatid disease, caused by the metacestode stage of the parasite.

Trypanocidal Activity of Aziridinyl Nitrobenzamide Prodrugs

The trypanocidal agents nifurtimox and benznidazole both function as prodrugs and must undergo enzyme-mediated activation, a reaction catalyzed by type I nitroreductase (NTR). In the search for new parasitic therapies, we have utilized this finding to investigate whether aziridinyl nitrobenzamide derivatives have activity against bloodstream-form Trypanosoma brucei and Trypanosoma cruzi amastigotes, parasite stages that replicate in the mammalian host. For T. cruzi drug screening, we generated trypanosomes that expressed the luciferase reporter gene and optimized a mammalian infection model in a 96-well plate format. A subset of compounds having a 5-(aziridin-1-yl)-2,4-dinitrobenzyl structure was shown to be metabolized by purified T. brucei NTR and when screened against both parasite life cycle stages displayed significant growth-inhibitory properties: the most potent compounds generated 50% inhibitory concentrations of <1 μM. The trypanocidal activity was shown to be NTR specific, since parasites overexpressing this enzyme were hypersensitive to the aziridinyl dinitrobenzyl agents. We conclude that members of the aziridinyl nitrobenzamide class of nitroheterocycles provide new lead structures that have the potential to treat trypanosomal infections.

Digital gene expression analysis of two life cycle stages of the human-infective parasite, Trypanosoma brucei gambiense reveals differentially expressed clusters of co-regulated genes

BackgroundThe evolutionarily ancient parasite, Trypanosoma brucei, is unusual in that the majority of its genes are regulated post-transcriptionally, leading to the suggestion that transcript abundance of most genes does not vary significantly between different life cycle stages despite the fact that the parasite undergoes substantial cellular remodelling and metabolic changes throughout its complex life cycle. To investigate this in the clinically relevant sub-species, Trypanosoma brucei gambiense, which is the causative agent of the fatal human disease African sleeping sickness, we have compared the transcriptome of two different life cycle stages, the potentially human-infective bloodstream forms with the non-human-infective procyclic stage using digital gene expression (DGE) analysis.ResultsOver eleven million unique tags were generated, producing expression data for 7360 genes, covering 81% of the genes in the genome. Compared to microarray analysis of the related T. b. brucei parasite, approximately 10 times more genes with a 2.5-fold change in expression levels were detected. The transcriptome analysis revealed the existence of several differentially expressed gene clusters within the genome, indicating that contiguous genes, presumably from the same polycistronic unit, are co-regulated either at the level of transcription or transcript stability.ConclusionsDGE analysis is extremely sensitive for detecting gene expression differences, revealing firstly that a far greater number of genes are stage-regulated than had previously been identified and secondly and more importantly, this analysis has revealed the existence of several differentially expressed clusters of genes present on what appears to be the same polycistronic units, a phenomenon which had not previously been observed in microarray studies. These differentially regulated clusters of genes are in addition to the previously identified RNA polymerase I polycistronic units of variant surface glycoproteins and procyclin expression sites, which encode the major surface proteins of the parasite. This raises a number of questions regarding the function and regulation of the gene clusters that clearly warrant further study.

Anti-schistosomal Intervention Targets Identified by Lifecycle Transcriptomic Analyses

BackgroundNovel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis.Methodology/Principal FindingsUtilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and/or adult transcripts with no known sequence similarity outside the Platyhelminthes), which were undetectable by the statistical comparisons.Conclusions/SignificanceCollectively, statistical and network-based exploratory analyses of transcriptomic datasets have led to a thorough characterisation of schistosome development. Information obtained from these experiments highlighted key transcriptional programs associated with lifecycle progression and identified numerous anti-schistosomal candidate molecules including G-protein coupled receptors, tetraspanins, Dyp-type peroxidases, fucosyltransferases, leishmanolysins and the netrin/netrin receptor complex.

Low-high season variation inPlasmodium falciparumerythrocyte binding antigen 175 (eba-175) allelic forms in malaria endemic area of Burkina Faso

To assess the impact of seasonal variation on the distribution of the eba-175 allelic forms in the area where malaria transmission is markedly seasonal. Blood samples were collected from 291 and 239 children under 5 years of age during the low and the high malaria transmission season, respectively, in four villages named Dawelgué, Kounda, Tanghin and Watenga of Saponé Health District, then screened for eba 175 F- and C- alleles by nested PCR analysis. F- alleles were more prevalent than C-alleles in the low [0.66 vs. 0.34 (P < 0.0001)] and high transmission season [0.67 vs. 0.33 (P < 0.0001)]. No significant seasonal variation was observed in the distribution of the two alleles. However, according to Sewall Wright rules, the population pairwise F(ST) values, between Dawelgué and Tanghin during the low transmission season (F(ST_) value = 0.10415, P-value = 0.0090 and during the high season (F(ST_)value = 0.08244, P-value < 0.00001), between Tanghin and Watenga during the low season (F(ST) value = 0.07414, P-value = 0.009) indicated a moderate but statistically significant genetic differentiation. Although there was a moderate but significant genetic differentiation between some study villages at different times of the year, this study result in the seasonal stability of eba-175 allele's distribution in the study area.

Fine mapping of Plasmodium falciparum ribosomal phosphoprotein PfP0 revealed sequences with highly specific binding activity to human red blood cells

The Plasmodium falciparum P0 ribosomal phosphoprotein (PfP0) was identified for the first time by screening a cDNA expression library of P. falciparum parasites with sera from malaria-immune individuals. Due to its localization on the surface of different parasite life-cycle stages (merozoites and gametocytes) and its recognition by invasion-blocking antibodies, PfP0 has been considered a potential malaria-vaccine component. In this study, 16 20-mer-long synthetic peptides spanning the entire PfP0 sequence were evaluated by means of receptor-ligand assays with human red blood cells (RBCs) in order to determine the role played by these peptides in the invasion process. Four RBC high-activity binding peptides (HABPs), located mostly toward the N-terminal region, were identified: HABP 33898 ((1)MAKLSKQQKKQMYIEKLSSL(20)), HABP 33900 ((41)ASVRKSLRGKATILMGKNTRY(60)), HABP 33901 ((61)IRTALKKNLQAVPQIEKLLPY (80)), and HABP 33906 ((161)LIKQGEKVTASSATLLRKFNY(180)). The binding pattern of HABPs 33898 and 33906 to enzyme-treated RBCs suggests receptors of protein nature for these two HABPs, one of which could correspond to a common 58-kDa RBC membrane protein, as indicated by results of cross-linking assays. Both HABPs exhibited high content of alpha-helical features and prevented P. falciparum merozoite invasion to RBCs in vitro by up to 91%. The invasion-blocking ability reported here for these PfP0 HABPs supports their inclusion in immunological studies with the aim of assessing their potential as candidates for a vaccine against P. falciparum malaria.

Plasmodium falciparum enolase: stage-specific expression and sub-cellular localization

BackgroundIn an earlier study, it was observed that the vaccination with Plasmodium falciparum enolase can confer partial protection against malaria in mice. Evidence has also build up to indicate that enolases may perform several non-glycolytic functions in pathogens. Investigating the stage-specific expression and sub-cellular localization of a protein may provide insights into its moonlighting functions.MethodsSub-cellular localization of P. falciparum enolase was examined using immunofluorescence assay, immuno-gold electron microscopy and western blotting.ResultsEnolase protein was detected at every stage in parasite life cycle examined. In asexual stages, enolase was predominantly (≥85–90%) present in soluble fraction, while in sexual stages it was mostly associated with particulate fraction. Apart from cytosol, enolase was found to be associated with nucleus, food vacuole, cytoskeleton and plasma membrane.ConclusionDiverse localization of enolase suggests that apart from catalyzing the conversion of 2-phosphoglycericacid into phosphoenolpyruvate in glycolysis, enolase may be involved in a host of other biological functions. For instance, enolase localized on the merozoite surface may be involved in red blood cell invasion; vacuolar enolase may be involved in food vacuole formation and/or development; nuclear enolase may play a role in transcription.

PiggyBac is an effective tool for functional analysis of the Plasmodium falciparumgenome

BackgroundMuch of the Plasmodium falciparum genome encodes hypothetical proteins with limited homology to other organisms. A lack of robust tools for genetic manipulation of the parasite limits functional analysis of these hypothetical proteins and other aspects of the Plasmodium genome. Transposon mutagenesis has been used widely to identify gene functions in many organisms and would be extremely valuable for functional analysis of the Plasmodium genome.ResultsIn this study, we investigated the lepidopteran transposon, piggyBac, as a molecular genetic tool for functional characterization of the Plasmodium falciparum genome. Through multiple transfections, we generated 177 unique P. falciparum mutant clones with mostly single piggyBac insertions in their genomes. Analysis of piggyBac insertion sites revealed random insertions into the P. falciparum genome, in regards to gene expression in parasite life cycle stages and functional categories. We further explored the possibility of forward genetic studies in P. falciparum with a phenotypic screen for attenuated growth, which identified several parasite genes and pathways critical for intra-erythrocytic development.ConclusionOur results clearly demonstrate that piggyBac is a novel, indispensable tool for forward functional genomics in P. falciparum that will help better understand parasite biology and accelerate drug and vaccine development.

The EG95 Antigen of Echinococcus spp. Contains Positively Selected Amino Acids, which May Influence Host Specificity and Vaccine Efficacy

Echinococcosis is a worldwide zoonotic parasitic disease of humans and various herbivorous domestic animals (intermediate hosts) transmitted by the contact with wild and domestic carnivores (definitive hosts), mainly foxes and dogs. Recently, a vaccine was developed showing high levels of protection against one parasite haplotype (G1) of Echinococcus granulosus, and its potential efficacy against distinct parasite variants or species is still unclear. Interestingly, the EG95 vaccine antigen is a secreted glycosylphosphatydilinositol (GPI)-anchored protein containing a fibronectin type III domain, which is ubiquitous in modular proteins involved in cell adhesion. EG95 is highly expressed in oncospheres, the parasite life cycle stage which actively invades the intermediate hosts. After amplifying and sequencing the complete CDS of 57 Echinococcus isolates belonging to 7 distinct species, we uncovered a large amount of genetic variability, which may influence protein folding. Two positively selected sites are outside the vaccine epitopes, but are predicted to alter protein conformation. Moreover, phylogenetic analyses indicate that EG95 isoform evolution is convergent with regard to the number of beta-sheets and alpha-helices. We conclude that having a variety of EG95 isoforms is adaptive for Echinococcus parasites, in terms of their ability to invade different hosts, and we propose that a mixture of isoforms could possibly maximize vaccine efficacy.

Trypanosoma cruzi bromodomain factor 2 (BDF2) binds to acetylated histones and is accumulated after UV irradiation

Histone tail post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation) regulate many cellular processes. Among these modifications, phosphorylation, methylation and acetylation have already been described in trypanosomatid histones. Bromodomains, together with chromodomains and histone-binding SANT domains, were proposed to be responsible for “histone code” reading. The Trypanosoma cruzi genome encodes four coding sequences (CDSs) that contain a bromodomain, named TcBDF1-4. Here we show that one of those, TcBDF2, is expressed in discrete regions inside the nucleus of all the parasite life cycle stages and binds H4 and H2A purified histones from T. cruzi. Immunolocalization experiments using both anti-histone H4 acetylated peptides and anti-TcBDF2 antibodies determined that TcBDF2 co-localizes with histone H4 acetylated at lysines K10 and K14. TcDBF2 and K10 acetylated H4 interaction was confirmed by co-immunoprecipitation. It is also shown that TcBDF2 was accumulated after UV irradiation of T. cruzi epimastigotes. These results suggest that TcBDF2 could be taking part in a chromatin remodelling complex in T. cruzi.

Real‐time imaging ofLeishmania mexicana‐infected early phagosomes: a study using primary macrophages generated from green fluorescent protein‐Rab5 transgenic mice

The small GTPase Rab5 is a key regulator of endosome/phagosome maturation and in intravesicular infections marks a phagosome stage at which decisions over pathogen replication or destruction are integrated. It is currently unclear whether Leishmania-infected phagosomes uniformly pass through a Rab5(+) stage on their intracellular path to compartments with late endosomal/early lysosomal characteristics. Differences in routes and final compartments could have consequences for accessibility to antileishmanial drugs. Here, we generated a unique gfp-rab5 transgenic mouse model to visualize Rab5 recruitment to early parasite-containing phagosomes in primary host cells. Using real-time fluorescence imaging of phagosomes carrying Leishmania mexicana, we determined that parasite-infested phagosomes follow a uniform sequence of transient Rab5 recruitment. Residence in Rab5(+) compartments was much shorter compared with phagosomes harboring latex beads. Furthermore, a comparative analysis of parasite life-cycle stages and mutants deficient in lpg1, the gene encoding the enzyme required for synthesis of the dominant surface lipophosphoglycan, indicated that parasite surface ligands and host cell receptors modulate pathogen residence times in Rab5(+) phagosomes, but, as far as tested, had no significant effect on intracellular L. mexicana survival or replication.

Characterization of rat bone marrow dendritic cells initially primed by Trichinella spiralis antigens

Pathogen-derived products have the capacity to induce maturation of bone marrow-derived dendritic cells (BMDCs)into populations of effectors cells that polarize Th cells toward Th1 or Th2 phenotype via different mechanisms. Since those mechanisms are not entirely clear for helminths, and almost completely unknown for Trichinella spiralis(TS), we started an investigation of the effects of TS antigens (four different antigens isolated from all three life-cycle stages of parasite)on maturation of BMDCs and their potential to present TS antigens. The expression of MHC class II, costimulatory molecules CD86, CD54, IL-10 and IL-12p70 cytokine production were measured after 2 days of BMDCs cultivation with TS antigens. While parasitic antigens did not significantly alter the expression of MHC II, most of them, except crude muscle larvae antigens, up-regulated the expression of costimulatory molecules. BMDCs, primed with all TS antigens, released increased amounts of IL-10 and decreased amounts of IL-12p70. BMDCs, primed with TS antigens, induced significant proliferation of syngeneic TS sensitized lymph nodes cells and also stimulated the production of IL-4 by T cells purified from of TS infected DA rats. The results indicate that TS stimulated BMDCs leads to the polarization of the immune response towards regulatory and Th2 type.

Plasmodium falciparum variant STEVOR antigens are expressed in merozoites and possibly associated with erythrocyte invasion

BackgroundPlasmodium falciparum STEVOR proteins, encoded by the multicopy stevor gene family have no known biological functions. Their expression and unique locations in different parasite life cycle stages evoke multiple functionalities. Their abundance and hypervariability support a role in antigenic variation.MethodsImmunoblotting of total parasite proteins with an anti-STEVOR antibody was used to identify variant antigens of this gene family and to follow changes in STEVOR expression in parasite populations panned on CSA or CD36 receptors. Immunofluorescence assays and immunoelectron microscopy were performed to study the subcellular localization of STEVOR proteins in different parasite stages. The capacity of the antibody to inhibit merozoite invasion of erythrocytes was assessed to determine whether STEVOR variants were involved in the invasion process.ResultsAntigenic variation of STEVORs at the protein level was observed in blood stage parasites. STEVOR variants were found to be present on the merozoite surface and in rhoptries. An insight into a participation in erythrocyte invasion was gained through an immunofluorescence analysis of a sequence of thin slides representing progressive steps in erythrocyte invasion. An interesting feature of the staining pattern was what appeared to be the release of STEVORs around the invading merozoites. Because the anti-STEVOR antibody did not inhibit invasion, the role of STEVORs in this process remains unknown.ConclusionThe localization of STEVOR proteins to the merozoite surface and the rhoptries together with its prevalence as a released component in the invading merozoite suggest a role of these antigens in adhesion and/or immune evasion in the erythrocyte invasion process. These observations would also justify STEVORs for undergoing antigenic variation. Even though a role in erythrocyte invasion remains speculative, an association of members of the STEVOR protein family with invasion-related events has been shown.

Antibody response against endogenous stages of an attenuated strain of Eimeria tenella

The application of attenuated vaccines for the prevention of chicken coccidiosis has increased exponentially in recent years. In Eimeria infections, protective immunity is thought to rely on a strong cell mediated response with antibodies supposedly playing a minor role. However, under certain conditions antibodies seem to be significant in protection. Furthermore, antibodies could be useful for monitoring natural exposure of flocks to Eimeria spp. and for monitoring the infectivity of live vaccines. Our objective was to investigate the chicken antibody response to the different parasite lifecycle stages following infection with an attenuated strain of Eimeria tenella. Western blotting analysis of parasite antigens prepared from the lining of caeca infected with the attenuated strain of E. tenella revealed two dominant antigens of 32 and 34 kDa, apparently associated with trophozoites and merozoites that were present at high concentrations between 84 and 132 h post-infection. When cryosections of caeca infected with E. tenella were probed with IgY purified from immune birds the most intense reaction was observed with the asexual stages. Western blotting analysis of proteins of purified sporozoites and third generation merozoites and absorption of stage-specific antibodies from sera suggested that a large proportion of antigens is shared by the two stages. The time-courses of the antibody response to sporozoite and merozoite antigens were similar but varied depending on the inoculation regime and the degree of oocyst recirculation.

Immunization against Egyptian &amp;lt;italic&amp;gt;Schistosoma mansoni&amp;lt;/italic&amp;gt; infection by multivalent DNA vaccine

The development of multivalent vaccines consisting of several antigens is a novel approach to creating broad-range protection against different parasite strains and parasite life cycle stages. We have previously confirmed that the schistosome Sm21.7 and SmFimbrin (SmFim) proteins could induce protection in mice. Therefore, this study aimed to construct the multivalent DNA vaccine Sm21.7-SmFim/pBudCE4.1 and evaluate its immune efficacy. The open reading frames of two Schistosoma mansoni genes, Sm21.7 and SmFim, were inserted into the eukaryotic expression plasmid pBudCE4.1 designed for the independent expression of two genes in mammalian cells. To evaluate the in vitro expression of the multivalent Sm21.7-SmFim/pBudCE4.1 DNA vaccine and its immunological effect in mice, the recombinant plasmid Sm21.7-SmFim/pBudCE4.1 was used to transfect 293T cells, and the expression of mRNA and proteins was examined using reverse transcription-polymerase chain reaction and Western blot analysis. Then the ability of Sm21.7-SmFim/pBudCE4.1 to protect against S. mansoni challenge infections was analyzed according to worm burden and egg reduction rates after vaccination of mice. Vaccinated mice showed a significant level of protection (56%), and a decrease in the number and size, and change in the cellular profile, of granulomas. Egg reduction in liver and intestine was 41.53% and 55.63%, respectively, as determined relative to mice that received the empty vector only. In addition to reductions in worm viability, worm fecundity and egg hatching ability were observed following challenge infection in the immunized group. Results showed that Sm21.7-SmFim/pBudCE4.1 could express Sm21.7 and SmFim mRNA and proteins. Enzyme-linked immunosorbent assay and Western blot analysis indicated that immunized mice generated specific immunoglobulin G against Sm21.7-SmFim/pBudCE4.1. These results suggest that vaccination with multivalent S. mansoni DNA vaccine (SmFim-Sm21.7/pBudCE4.1) not only induces a significant reduction in worm and egg burdens, but also significantly reduces the size of egg granulomas. In summary, the multivalent vaccine stimulated specific immunity with a significant level of protection and has anti-pathological effect.

Monitoring Plasmodium falciparum growth and development by UV flow cytometry using an optimized Hoechst‐thiazole orange staining strategy

The complex life cycle of Plasmodium falciparum (Pf) makes it difficult to limit infections and reduce the risk of severe malaria. Improved understanding of Pf blood-stage growth and development would provide new opportunities to evaluate and interfere with successful completion of the parasite's life cycle. Cultured blood stage Pf was incubated with Hoechst 33342 (HO) and thiazole orange (TO) to stain DNA and total nucleic acids, respectively. Correlated HO and TO fluorescence emissions were then measured by flow cytometry. Complex bivariate data patterns were analyzed by manual cluster gating to quantify parasite life cycle stages. The permutations of viable staining with both reagents were tested for optimal detection of parasitized RBC (pRBC). Pf cultures were exposed to HO and TO simultaneously to achieve optimal staining of pRBC and consistent quantification of early and late stages of the replicative cycle (rings through schizonts). Staining of Pf nucleic acids allows for analysis of parasite development in the absence of fixatives, lysis, or radioactivity to enable examination of erythrocytes from parasite invasion through schizont rupture using sensitive and rapid assay procedures. Investigation of the mechanisms by which anti-malarial drugs and antibodies act against different Pf lifecycle stages will be aided by this cytometric strategy.

A combined transcriptome and proteome survey of malaria parasite liver stages

For 50 years since their discovery, the malaria parasite liver stages (LS) have been difficult to analyze, impeding their utilization as a critical target for antiinfection vaccines and drugs. We have undertaken a comprehensive transcriptome analysis in combination with a proteomic survey of LS. Green fluorescent protein-tagged Plasmodium yoelii (PyGFP) was used to efficiently isolate LS-infected hepatocytes from the rodent host. Genome-wide LS gene expression was profiled and compared with other parasite life cycle stages. The analysis revealed approximately 2,000 genes active during LS development, and proteomic analysis identified 816 proteins. A subset of proteins appeared to be expressed in LS only. The data revealed exported parasite proteins and LS metabolic pathways including expression of FASII pathway enzymes. The FASII inhibitor hexachlorophene and the antibiotics, tetracycline and rifampicin, that target the apicoplast inhibited LS development, identifying FASII and other pathways localized in the apicoplast as potential drug targets to prevent malaria infection.